Abstract
Low density lipoprotein cholesteryl [14C]oleate (LDL-[14C]CO) was used as a tool to label lysosomes with cholesteryl [14C]oleate (CO) and to follow subsequently the metabolic processing of oleic acid released by acid lipase. Liberated [14C]oleate was incorporated into glycerolipids, mainly into phosphatidylcholine. Incubations in the presence of various concentrations of exogenously added oleic acid and double label experiments showed that oleic acid derived from lysosomal degradation of CO and exogenously added oleic acid distributed in a similar fashion among triacylglycerol and various phospholipids. To further study the metabolism of LDL-derived oleic acid, experiments were performed in which fibroblasts were prelabeled with LDL-[14C]CO. The subsequent processing of lysosome-derived oleic acid was followed with time without LDL-[14C]CO in the medium. From these experiments it became clear that apart from the esterification into glycerolipids a substantial part of lysosome-derived oleic acid was released into the medium. The efflux of oleic acid into the medium preceded the incorporation into glycerolipids, was dependent on the composition of the extracellular medium, and was energy-independent. Our data are compatible with a mechanism in which lysosome-derived fatty acids are transported to the plasma membrane prior to transport to endoplasmic reticulum for esterification. Intra- and extra-cellular factors influence the distribution of lysosome-derived oleic acid among cells and medium.
Highlights
Low density lipoprotein cholesteryl ["C] oleate (LDL-['4C]CO)was used as a tool to label lysosomes with cholesteryl ['4C]oleate (CO) and to follow subsequently the metabolic processing of oleic acid released by acid lipase
In contrast to control fibroblasts, in fibroblasts derived from a patient with Wolman disease CO was not degraded and all cell-associated radioactivity was found to be present in cholesteryl ester,This confirms that low density lipoprotein (LDL)[I4C]GO is taken up via the LDL-receptor pathway and is exclusively degraded in lysosomes by acid lipase
We first showed that the LDL labeled with ['4C]chole~teryol leate by the use of plasma lipid transfer protein is taken up by the LDL receptor and that, subsequently, cholesteryl ester is exclusively catabolized by lysosomal acid lipase
Summary
Low density lipoprotein cholesteryl ["C] oleate (LDL-['4C]CO)was used as a tool to label lysosomes with cholesteryl ['4C]oleate (CO) and to follow subsequently the metabolic processing of oleic acid released by acid lipase. The subsequent processing of lysosomederived oleic acid was followed with time without LDL-[14C]C0in the medium From these experiments it became clear that apart from the esterification into glycerolipids a substantial part of lysosome-derivedoleic acid was released into the medium. The fact that cholesterol, derived from low density lipoprotein (LDL), accumulates inside lysosomes in Niemann-Pick type C [4, 5] is of interest and suggests that at least o n e of the lipid e n d products of lysosomal catabolism is not freely transportable across the lysosomal membrane. Inherited defects in individual steps of lysosomal degradation of macromolecu~esresult in lysosomal storage of muco-
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